As critical as O2 is for maintaining cellular, and ultimately organismal homeostasis, there is little specific information that quantifies the relationship between intracellular O2 levels and cell function. The objective of this project is to continue experimentation designed to determine the O2 dependence of cell respiration, metabolism, and function in isolated single skeletal muscle fibers. Specifically, we will test the general hypotheses single skeletal muscle fibers. Specifically, we will test the general hypotheses that cellular function is affected, both directly and indirectly, by intracellular [O2] levels considerably above those considered rate limiting. for isolated mitochondria. Single, membrane intact, skeletal muscle fibers will be isolated from Xenopus laevis and placed into a chamber in which the extracellular milieu can be precisely controlled, metabolic and respiratory rate can be varied by stimulation, Q2 uptake measured, and optical imaging can be conducted. To test our primary hypothesis, intracellular P02 will be measured (using a phosphorescence lifetime technique) under varied conditions and the relationships between cell P02 and respiratory, glycolytic rate, the regulators of oxidative phosphorylation, and the regulation of contractile function will be determined. The relationship between mitochondrial density (and distribution) and the processes listed above will be examined in three different Xenopus fiber types. In addition, experiments are proposed that test hypotheses concerning the importance of myoglobin in intracellular O2 transport, potential intracellular O2 sensors, causes of cell damage related to inadequate oxygenation, and energy sources for contractile work with altered oxygenation. By utilizing an isolated single skeletal muscle cell, intrinsic properties of the working cell can be investigated without confounding factors of microcirculation and fiber type heterogeneities. The originality and significance of the proposed experiments residue in the integration of several established and new techniques to investigate cell function as it is affected by [O2], and the unique research expertise of the personnel assembled in this program project application provides a singular opportunity to study these processes. The proposed studies will provide valuable information defining the O2 dependence of cellular function; which has direct implications concerning cell, organ, and organismal health, particularly during disease states involving O2 deprivation.